1. Field of the Invention
The present invention relates generally to the fields of microbiology, bacteriology and molecular biology. More specifically, the present invention relates to the molecular cloning and characterization of the Ehrlichia chaffeensis 28 kD outer membrane protein multigene family.
2. Description of the Related Art
Ehrlichia are small, obligatory intracellular, gram negative bacteria which reside in endosomes inside host cells. Ehrlichiae usually cause persistent infection in their natural animal hosts (Andrew and Norval, 1989, Breitschwerdt et al., 1998, Dawson et al., 1994, Dawson and Ewing, 1992, Harrus et al., 1998, Telford et al., 1996). Persistent or prolonged Ehrlichia infections in human hosts have also been documented (Dumler et al., 1993, Dumler and Bakken, 1996, Horowitz, et al., 1998, Roland et al. 1994). The persistent infection may be caused by the antigenic variation of the Ehrlichia omp-2 and p28 outer membrane protein family due to differential expression or recombination of the msp-2 multigene family (Palmer et al., 1994, Palmer et al., 1998) or the p28 multigene family (Ohashi et al., 1998b, Reddy et al., 1998, Yu et al., 1999b).
The omp-2 and p28 are homologous gene families coding for outer membrane proteins. The msp-2 multigene family has been identified in A. marginale (Palmer et al., 1994), A. ovina (Palmer et al., 1998), and the human granulocytotropic ehrlichiosis agent (Ijdo et al.,1998, Murphy et al.,1998). The p28 multigene family has been found in E. canis group ehrlichiae including E. canis, E. chaffeensis, and E. muris (McBride et al., 1999a, 1999b, Ohashi et al., 1998a, 1998b, Reddy et al., 1998, Yu et al., 1999a, 1999b). The map-1 multigene family found in Cowdria ruminantium is more closely related to the p28 multigene family than to the msp-2 multigene family, both in sequence similarity and gene organization (Sulsona et al., 1999, van Vliet et al., 1994). The msp-2 genes are dispersed in the genome whereas the p28/map-1 genes are located in a single locus.
To elucidate the mechanism of the host immune avoidance involving the multigene family, the critical questions that remain to be answered are how many genes are present in each multigene family and which genes are silent or active. E. chaffeensis is the pathogen of an emerging disease, human monocytotropic ehrlichiosis. Recent studies have found seven homologous polymorphic p28 genes in E. chaffeensis which encode proteins from 28 to 30-kDa (Ohashi et al., 1998b, Reddy et al., 1998). The seven sequenced p28 genes were located in three loci of the E. chaffeensis genome. The first locus, omp-1 contained six p28 genes. One gene was partially sequenced (omp1-a) and five genes were completely sequenced (omp-1b, -1c, -1d, -1e, and -1f) (Ohashi et al., 1998b). The second locus contained a single p28 gene (Ohashi et al., 1998b, Yu et al., 1999b). The third locus contained five p28 genes (ORF 1 to 5). The first four open reading frames overlapped with the DNA sequences from omp-1 c to omp-1f and the fifth open reading frame overlapped with the single gene in the second locus. Therefore, the three loci could be assembled into a single locus (Reddy et al., 1998).
The prior art is deficient in the lack of the knowledge of many of the sequences of the genes in the p28 multigene family of E. chaffeensis. The present invention fulfills this long-standing need and desire in the art.